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We conducted an observational study aimed at analyzing the presence of IgA and IgG antibodies against all the structural proteins of SARS-CoV-2 in breast milk and serum samples derived from 30 vaccinated/naturally infected breastfeeding mothers. Samples were collected from October to December 2022. The inclusion criteria were: COVID-19 vaccination (at least one dose) or natural infection by SARS-CoV-2, breastfeeding (exclusive or mixed), and a number of children (single child or more). A survey was used for the collection of clinical and epidemiological data. It included: age; diagnosis of COVID-19 via RT-PCR, antigen, or clinical/unknown (not reported by the patient); serological status; symptomatology (fever, headache, loss of smell, loss of taste, dyspnea, chest pain, cough, sore throat, burning eyes, congested nose, muscle pain, joint pain, fatigue, chills, vomit, and diarrhea); number of infections; vaccine doses; and type of breastfeeding.

As an antigen, we used recombinant proteins derived from the SARS-CoV-2 (S1 domain, RBD domain; Receptor Binding Domain, N, M, and E), and purification was performed as previously described [18]. Microtiter plates (Sigma-Aldrich, St. Louis, MO, USA) were coated with 100 µL/well of each antigen (S1 domain, RBD domain, N, M, and E) to a final concentration of 0.1 µg/mL in a coating buffer (50 mM Na₂CO₃/NaHCO₃, pH 9.6). The plates were incubated for one hour at 37 °C and then blocked for 40 min at 37 °C with 200 µL/well of 5% skimmed milk diluted in phosphate-buffered saline (PBS)-Tween 20 (0.05%). For the primary antibody, 100 µL/well of serum samples (1:50 diluted in PBS) or breast milk (direct) by duplicate were incubated for one hour at 37 °C. Finally, plates were incubated with 100 µL/well of monoclonal anti-human IgA (Sigma-Aldrich; 1:500 dilution) and IgG (Sigma-Aldrich; 1:1500 dilution, St. Louis, MO, USA) coupled to horseradish peroxidase (HRP) for one hour at 37 °C. After every step, the plates were washed thrice with 200 μL/well of PBS-Tween 20 0.05% for 5 min. The enzymatic reaction was developed using o-phenylenediamine dihydrochloride (Sigma-Aldrich) and stopped using 2 N H₂SO₄. The optical density (OD) was measured at 492 nm using a microplate reader. Samples with OD > 0.300 were considered positive for both antibodies.

For IgG subclass evaluation, antigens were fixed and plates were blocked as described above. Serum samples were prepared (1:25 diluted in PBS), and each sample was tested by duplicate to each antigen and subclass. Plates were incubated for 30 min at 37 °C. Later, monoclonal antibodies against each subclass: IgG1 (1:500; Sigma-Aldrich Cat# B6775), IgG2 (1:25,000; Sigma-Aldrich Cat#B3398), IgG3 (1:500; Sigma-Aldrich Cat#B3523), and IgG4 (1:1,000; Sigma-Aldrich Cat#B3648) were added for 45 min at 37 °C. As a secondary antibody, anti-mouse IgG (H+L) coupled to HRP (Invitrogen Cat# 62-6520) was diluted 1:11,000 for 45 min at 37 °C. After each step, plates were washed for 7 min with PBS-Tween 20 0.05%. Finally, the enzymatic reaction was developed as above. The cut-off value for each antibody was: IgG1 ≥ 0.070, IgG2 ≥ 0.175, IgG3 ≥ 0.112, and IgG4 ≥ 0.100, as we previously described (manuscript under review).

The natural infection or vaccination of COVID-19 during pregnancy or breastfeeding is vital for developing immunity in the newborn. We reported a high prevalence of IgA antibodies against all the structural proteins of SARS-CoV-2 in breast milk (76.67–100%) and a moderate prevalence of IgG (23.3–60%) and IgA (10–36.67%) in serum samples. Our results suggest that breast milk contains antibodies capable of recognizing and neutralizing SARS-CoV-2 to confer immunity to the newborn. In the bloodstream, IgG is the most dominant, and its biological functions—associated with the specific presence of IgG1, IgG2, and IgG4 subclasses—are neutralization, complement activation, and phagocytosis, among others. It is imperative to perform evaluations focused on the presence of total IgG and IgA antibodies against the S and N proteins. However, we must also include M, E, and IgG subclasses to fully understand the immune response developed against SARS-CoV-2.